ER-positive Breast Cancer Cell Lines Research Articles

Adipocyte-conditioned medium induces tamoxifen resistance by activating PI3K/Akt/mTOR pathway in estrogen receptor-positive breast cancer cells.

Resistance to endocrine therapy is a major clinical challenge in estrogen receptor (ER)-positive breast cancer. Obesity is associated with the clinical response to ER-positive breast cancers; however, the mechanism underlying obesity-induced resistance to endocrine therapy in ER-positive breast cancers remains unclear. In this study, we investigated the molecular mechanisms underlying obesity-induced resistance to tamoxifen (TAM), an anti-estrogen agent, in the ER-positive breast cancer cell line MCF-7 using differentiated adipocyte-conditioned medium (D-CM). Treatment of the cells with D-CM promoted TAM resistance by reducing TAM-induced apoptosis. The expression levels of the ERα target genes were higher in D-CM-treated cells than those in untreated ones. In contrast, when the cells were cultured in the presence of TAM, the expression levels were decreased, with or without D-CM. Moreover, the expression of the markers for cancer stem-like cells (CSCs) and mammosphere formation was enhanced by co-treating with D-CM and TAM, compared with TAM alone. The phosphatidylinositol-3-kinase (PI3K)/Akt/mammalian target of rapamycin (mTOR) pathway was activated in MCF-7 cells by D-CM treatment, even in the presence of TAM. Inhibition of the PI3K/Akt/mTOR pathway decreased the expression levels of the CSC markers, suppressed mammosphere formation, and resensitized to TAM via inducing apoptosis in D-CM-treated cells. These results indicate that the conditioned medium of differentiated adipocytes promoted TAM resistance by inducing the CSC phenotype through activation of the PI3K/Akt/mTOR pathway in ER-positive breast cancer cells. Thus, the PI3K/Akt/mTOR pathway may be a therapeutic target in obese patients with ER-positive breast cancers.

Targeting Cholesterol Biosynthesis with Statins Synergizes with AKT Inhibitors in Triple-Negative Breast Cancer.

Triple-negative breast cancer (TNBC) is responsible for a disproportionate number of breast cancer patient deaths due to extensive molecular heterogeneity, high recurrence rates, and lack of targeted therapies. Dysregulation of the phosphoinositide 3-kinase (PI3K)/AKT pathway occurs in approximately 50% of TNBC patients. Here, we performed a genome-wide CRISPR/Cas9 screen with PI3Kα and AKT inhibitors to find targetable synthetic lethalities in TNBC. Cholesterol homeostasis was identified as a collateral vulnerability with AKT inhibition. Disruption of cholesterol homeostasis with pitavastatin synergized with AKT inhibition to induce TNBC cytotoxicity in vitro in mouse TNBC xenografts and in patient-derived estrogen receptor (ER)-negative breast cancer organoids. Neither ER-positive breast cancer cell lines nor ER-positive organoids were sensitive to combined AKT inhibitor and pitavastatin. Mechanistically, TNBC cells showed impaired sterol regulatory element-binding protein 2 (SREBP-2) activation in response to single-agent or combination treatment with AKT inhibitor and pitavastatin, which was rescued by inhibition of the cholesterol-trafficking protein Niemann-Pick C1 (NPC1). NPC1 loss caused lysosomal cholesterol accumulation, decreased endoplasmic reticulum cholesterol levels, and promoted SREBP-2 activation. Taken together, these data identify a TNBC-specific vulnerability to the combination of AKT inhibitors and pitavastatin mediated by dysregulated cholesterol trafficking. These findings support combining AKT inhibitors with pitavastatin as a therapeutic modality in TNBC. Significance: Two FDA-approved compounds, AKT inhibitors and pitavastatin, synergize to induce cell death in triple-negative breast cancer, motivating evaluation of the efficacy of this combination in clinical trials.

P53 Status Influences the Anti-proliferative Effect Induced by IFITM1 Inhibition in Estrogen Receptor-positive Breast Cancer Cells.

Interferon-induced trans-membrane protein 1 (IFITM1) is known to be involved in breast cancer progression. We aimed to investigate its role in estrogen receptor (ER)-positive breast cancer cells with wild-type p53 and tamoxifen-resistant breast cancer cells. The ER-positive breast cancer cell lines, MCF-7 with wild-type p53 and T47D with mutant p53, were used. We established an MCF-7-derived tamoxifen-resistant cell line (TamR) by long-term culture of MCF-7 cells with 4-hydroxytamoxifen. IFITM1 inhibition in MCF-7 cells significantly decreased cell growth and migration. MCF-7 cells with suppression of IFITM1 using siRNA or ruxolitinib showed reduced cell viability after tamoxifen treatment compared with that in the control MCF-7 cells. Unexpectedly, mRNA and protein levels of IFITM1 were decreased in TamR cells compared with those in MCF-7 cells. TamR cells with suppression of IFITM1 using siRNA or ruxolitinib showed no change in cell viability after treatment with tamoxifen. P53 knockdown using siRNA reduced the mRNA levels of IRF9 and increased mRNA and protein levels of SOCS3 in MCF-7 cells, suggesting that loss or mutation of p53 can affect the induction of IFITM1 via the JAK/STAT signaling pathway in breast cancer. Furthermore, MCF-7 cells with p53 knockdown using siRNA showed no decrease in cell viability after tamoxifen treatment or IFITM1 inhibition, indicating that p53 status may be important for cell death after tamoxifen treatment or IFITM1 inhibition. IFITM1 inhibition may enhance the sensitivity to tamoxifen based on p53-dependent enhancement of IFN signaling in wild-type p53, ER-positive breast cancer cells.

Apoptotic effect of melatonin on ER-positive breast cancer cell lines: ADGRL4 gene expression and promoter methylation.

Breast cancer (BC) is the second most common malignancy worldwide. ADGRL4, as a modulator of angiogenesis, undergoes various epigenetic modifications affecting its biological functions. In this study, we aimed to assess ADGRL4 promoter methylation status and its expression levels in primary breast tumors and to evaluate its potency as a plausible prognostic biomarker in BC. Furthermore, we evaluated the effect of melatonin on ADGRL4 expression and viability of BC cells in vitro. One hundred breast tumor tissue samples and adjacent non-tumor tissues were collected, followed by DNA isolation, bisulfite conversion, qRT-PCR, qMSP assay, and immunoblotting. In addition, four BC cell lines were treated with melatonin and subjected to ADGRL4 expression analysis and apoptosis assay. We found a significant correlation between ADGRL4 expression levels and HER2 status and stage of disease (P < 0.05). We observed a substantial attenuation in ADGRL4 promoter methylation in tumor samples compared to marginal non-tumor samples. A significantly lower expression of ADGRL4 was detected in two BC cell lines in the presence of melatonin. MCF-7 and BT474 melatonin-treated cell lines showed a significantly higher number of apoptotic cells than non-treated cells (P < 0.0001). Based on the receiver operating characteristic (ROC) curve analysis, ADGRL4 expression and ADGRL4 promoter methylation status showed moderate prognostic value. We found that melatonin has anti-cancer effects on BC cells. In addition, ADGRL4 expression can potentially be used as a prognostic biomarker in BC.

Synthesis and estrogenic activity of BODIPY-labeled estradiol conjugates

Novel BODIPY–estradiol conjugates have been synthesized by selecting position C-3-O for labeling. The conjugation strategy was based on Cu(I)-catalyzed azide–alkyne cycloaddition (CuAAC) or etherification. Estradiol derivatives used as azide partners bearing an ω-azidoalkyl function through C4–C8-long linkers have been prepared. CuAAC reactions of estradiol azides with BODIPY alkyne furnished fluorescent 3-O-labeled conjugates bearing the triazole ring as a coupling moiety. Williamson etherifications of 3-O-(ω-bromoalkyl)-17β-estradiol derivatives with BODIPY-OH resulted in labeled conjugates connected with an ether moiety. Interactions of the conjugates with estrogen receptor (ER) were investigated using molecular docking calculations in comparison with estradiol. The conjugates occupied both the classical and alternative binding sites on human ERα, with slightly lower binding affinity to references estradiol and diethystilbestrol. All compounds have displayed reasonable estrogenic activity. They increased the proliferation of ER-positive breast cancer cell line MCF7 contrary to ER-negative SKBR-3 cell line. The most potent compound 13a induced the transcriptional activity of ER in dose-dependent manner in dual luciferase recombinant reporter model and increased progesterone receptor's expression, proving the retained estrogenic activity. The fluorescence of candidate compound 13a co-localised with the ERα. The newly synthesized labeled compounds might serve as good starting point for further development of fluorescent probes for modern biological applications. In addition to studying steroid uptake and transport in cells, e.g. in the processes of biodegradation of estrogen-hormones micropollutants, they could also be utilized in examination of estrogen-binding proteins.

257 (PB-073) Poster - Metronomic treatment significantly enhanced cytotoxicity in palbociclib-resistant ER-positive breast cancer cell lines

257 (PB-073) Poster - Metronomic treatment significantly enhanced cytotoxicity in palbociclib-resistant ER-positive breast cancer cell lines

Estrogen Receptor Is Required for Metformin-Induced Apoptosis in Breast Cancer Cells Under Hyperglycemic Conditions.

About 25% to 30% of estrogen receptor (ER)-positive breast cancer patients develop resistance to endocrine therapy. Human epidermal growth factor receptor 2 (HER2) has been shown to cooperate with several growth factors that regulate cellular energy metabolism, including the insulin-like growth factor 1 receptor (IGF-1R). As the first-line therapy for type 2 diabetes mellitus (T2DM) patients, metformin is widely known to inhibit the metabolic reprogramming of cancer cells. This study aims to investigate metformin's efficacy in inhibiting endocrine resistance related to genes regulating energy metabolism in both ER-positive and ER-negative breast cancer cell lines under hyperglycemic conditions. MDA-MB-361 (ER-positive, HER2-positive) and SKBR3 (ER-negative, HER2-positive) cancer cell lines were used to represent ER status. Cell viability and cell survival rate were measured using the colorimetric assay of Cell Counting Kit-8. All mRNA levels were quantified using real-time quantitative polymerase chain reaction preceded by reverse transcription. A P value of <.05 was considered statistically significant. Unlike MDA-MB-361, SKBR3 were found to acquire resistance upon metformin treatment in hyperglycemic conditions. Moreover, the mRNA expression of IGF-1R and its downstream signaling, such as the mammalian target of rapamycin (mTOR), was not affected by metformin. Meanwhile, the mRNA expression level of ribosomal S6 kinase 1 (S6K1) was upregulated, whereas forkhead box O1 (FOXO1) was downregulated after metformin treatment in hyperglycemic conditions. This preliminary study suggests that an alternative pathway of metformin resistance may exist in the absence of ERα. Therefore, relying solely on metformin may be inadequate to inhibit the aggressiveness of breast cancer cells.

Paradise Tree Extract (Simarouba glauca) Selectively Induces Cell Death, Enhances Efficacy of Common Chemotherapeutics and Reduces Their Toxicity in In-Vitro and In-Vivo Models of Breast Cancer

Background: Although chemotherapeutics have proven to be effective in treating metastatic breast cancer, their limited target selectivity has resulted in adverse side effects, rendering them unsuitable for long-term usage. Alternatively, certain natural extracts provide a promising strategy to selectively target cancer while being safe to consume. Specifically, paradise tree (Simarouba glauca) has shown potential anti-tumour activity; however, its efficacy against cancer, mechanism of action, and interaction with standard chemotherapies have not been investigated. Method: We have demonstrated the anti-tumour activity of ethanolic paradise tree extract (PTE) in triple-negative and ER-positive breast cancer cell lines and its interaction with chemotherapeutics when used in combination. The anti-tumour efficacy of PTE was evaluated through the expression of apoptotic biochemical markers as well as changes in cell morphology. For mechanistic studies, fluorogenic dyes were used to quantify reactive oxygen species production and mitochondrial membrane potential destabilization. Results: Our results have shown that PTE selectively triggers apoptosis in breast cancer cells while having limited effects on noncancerous cells. Importantly, we have found that PTE enhances the anti-tumour efficacy of chemotherapeutics, cisplatin and Taxol, when given in combination, while reducing their toxicity in noncancerous cells. Furthermore, PTE inhibits growth of human tumour xenografts in immunocompromised mice. Importantly, PTE in combination with Taxol and cisplatin had the best anti-tumour effect. Conclusion: Our findings suggest that PTE could be a safe and effective treatment for breast cancer. Most importantly, as a supplement to chemotherapeutic regimens, it could enhance anti-tumour effects and reduce chemo-related toxicity.

Abstract P2-25-03: β-arrestin1, a potential tumour suppressor in breast cancer is downregulated in PERK/ATF4-dependent manner

Abstract Introduction: β-Arrestin1 (ARRB1) belongs to the arrestin family originally identified as a multifunctional adaptor protein that negatively regulates the desensitization and internalization of G-protein-coupled receptors. Recent studies have indicated that ARRB1 can form multiprotein complexes with transcription regulators, thereby indirectly inhibiting the function of some transcription factors, e.g., NF-kB, and promoting the activity of others, e.g., p53. Further, arrestins function as multifunctional adaptors in many signalling pathways, such as the Hedgehog (Hh), Wingless, Notch, and transforming growth factor-β (TGF-β) pathways. Expression of ARRB1 is downregulated in TNBC patients and ARRB1 expression level is inversely correlated with the histological grade of the breast cancer and positively associated with TNBC patient survival, suggestive of a tumour-suppressive function of ARRB1 in breast cancer. Loss of ARRB1 expression is associated with a poor prognosis for non-small cell lung cancer patients. However, the mechanisms regulating the expression of ARRB1 in human cancers remain unclear. We hypothesized that metabolic endoplasmic reticulum (EnR) stressors including low glucose and hypoxia that activate an unfolded protein response (UPR) in tumour cells might regulate the expression of ARRB1 in human cancers. Methods: Expression of ARRB1 was determined by a combination of real-time RT-PCR, western blotting along with promoter reporter assays. Computational target prediction analysis was done to identify miRNAs targeting ARRB1 as well as transcription factors regulating ARRB1 expression. ARRB1overexpressing breast cancer clones were generated by lentiviral transduction to investigate its functional role in tumour progression and drug sensitivity. Results: We observed that ARRB1 expression was significantly reduced in several cancers including breast tumours as compared to normal tissue and decreased expression of ARRB1 was associated with poor prognosis in Luminal A subtype. We found significant downregulation of ARRB1 mRNA and protein during conditions of EnR stress in multiple ER-positive breast cancer cell lines. Using PERK, ATF6 and XBP1 knockdown sub-clones of MCF7 we observed that PERK signalling was required for the downregulation of ARRB1. ATF4 and miR-204/211 downstream of PERK were identified as key regulators of ARRB1 expression. Conclusion: Our results show that ATF4 and miR-204/211 downstream of the PERK signalling pathway repress ARRB1 expression in breast cancer cells. Our results suggest that the downregulation of ARRB1 by EnR stressors in the tumour microenvironment may contribute to breast cancer progression. Citation Format: Afrin Sultana, Ananya Gupta, Sanjeev Gupta. β-arrestin1, a potential tumour suppressor in breast cancer is downregulated in PERK/ATF4-dependent manner [abstract]. In: Proceedings of the 2022 San Antonio Breast Cancer Symposium; 2022 Dec 6-10; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2023;83(5 Suppl):Abstract nr P2-25-03.

Abstract P3-11-06: Chromatin landscape analysis based on ATAC-seq and RNA- seq reveals that GRHL2 is a novel key transcription factor for endocrine therapy resistance

Abstract Current data using gene expression and DNA modification profiles yield limited information for evaluating the potential resistance to therapies. Recent studies show that the chromatin landscape is dynamic and defines cell identity and disease status. Thus, changes in open chromatin sites could provide important information on disease progression and identify potential therapeutic targets. The majority of breast cancers express estrogen receptor (ER) and initially respond to endocrine therapies blocking ER activity. However, endocrine therapy resistance (ETR) occurs de novo or follows an initial response. Recent studies have identified new drivers of ETR, such as mTOR and CDK4/6. Therapy with these inhibitors in combination with endocrine therapy have improved patient survival, but the molecular mechanism of ETR is not fully understood and additional therapeutic options for ETR are needed. To investigate the molecular mechanisms of ETR, we established in vitro Long-Term Estrogen Deprivation (LTED) model using multiple human ER-positive breast cancer cell lines. These cells showed differential time-dependent patterns of ESR1 expression upon LTED with ESR1 upregulation in some cell lines and downregulation in others. LTEDs with ESR1 upregulation were sensitive to fulvestrant, while the others were resistant. We analyzed time-course of chromatin landscape transitions during acquisition of ETR in ESR1 up- and downregulated LTEDs cells by Assay for Transposase Accessible Chromatin (ATAC-seq) combined with RNA-seq. Because transcription factors play a primary key role in gene expression and in cell-fate changes including response to therapies, we performed bioinformatic analysis to uncover differential open chromatin sites, select enhancer signatures and identify digital footprints of transcription factors. Unlike ChIP-seq, which identifies binding sites for a known transcription factor, ATAC-seq permits analysis of all sites potentially accessible to the transcriptional machinery and predict occupancy by ~700 different transcription factors at once. With these algorithms, we found that Grainyhead-like 2 (GRHL2) is associated with development of both ESR1 overexpressed and downregulated types of LTEDs. Furthermore, RNA-seq analysis showed that GRHL2 target genes are upregulated in LTEDs. Furthermore, in the METABRIC database, high GRHL2 expression is significantly associated with poor outcome in ER+ breast cancer patients treated with endocrine therapy. Taken together, these data suggest that GRHL2 may be a novel important transcription factor in endocrine therapy resistant breast cancer regardless of fulvestrant sensitivity. Our studies provide evidence that chromatin landscape analysis, coupled with the transcription factor network algorithm, is capable of identifying novel biomarkers or therapeutic targets, This approach will expand the range of translational research as it is applicable to many cancers and diseases. Citation Format: Saori Fujiwara, Songjoon Baek, Kaustubh Wagh, Diana Stavreva, Lyuba Varticovski, Gordon Hager. Chromatin landscape analysis based on ATAC-seq and RNA- seq reveals that GRHL2 is a novel key transcription factor for endocrine therapy resistance [abstract]. In: Proceedings of the 2022 San Antonio Breast Cancer Symposium; 2022 Dec 6-10; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2023;83(5 Suppl):Abstract nr P3-11-06.

Abstract P5-13-06: Overexpression of somatic mutation in NSDHL promotes breast cancer cell migration and tumor growth

Abstract Background: We had identified somatic mutations of the NAD(P) Dependent Steroid Dehydrogenase-Like (NSDHL) gene from breast tumors of patients with distant metastasis using whole-exome sequencing. This study aimed to investigate the function of NSDHL mutants in breast cancer. Methods: ER-positive and tamoxifen-resistant human breast cancer cell line (ZR75-1) and mouse macrophage cell line (RAW264,7) were used. GFP-tagged NSDHL mutants were generated by subcloning the mutant NDHL into a lentiviral vector yielding an in-frame fusion of 3′ to GFP. ZR75-1 cells expressing the GFP-tagged NSDHL mutants were selected by puromycin with subsequent FACS analysis. The following experiments were performed: western blot, immunofluorescence staining, qRT-PCR, CellTiter-Glo assay, cell cycle assay, transwell migration assay, wound healing assay, tumor spheroid formation assay, and total cellular cholesterol measurement. An orthotopic breast tumor mouse model by injection of ZR75.1 cells into mammary gland fat pad was used in vivo. Results: Four novel NSDHL somatic mutations were discovered in the breast tumor tissues of patients. While total cholesterol levels in NSDHL mutant-transduced cells did not significantly increase, they were notably higher in wild-type NSDHL-transduced cells than in parent ZR75-1 cells. When compared to the parent and wild-type NSDHL-transduced cells, there was a minor increase in growth rate and a large increase in migratory capacities in NSDHL mutant-transduced cells (P&amp;lt; 0.05). The size of tumor spheroids is significantly larger in NSDHL mutant-transduced cells than in wild-type NSDHL-transduced cells. Both EGFR expression and epithelial to mesenchymal transition (EMT) markers were higher in NSDHL mutant-transduced cells than in parent and wild-type NSDHL-transduced cells. The NSDHL mutant-transduced cells’ conditioned media promoted the migratory ability and M2 polarization of RAW264.7 cells. In an orthotopic xenograft mouse model, NSDHL mutant-transduced cells were shown to promote tumor growth better than parent and wild-type NSDHL-transduced cells, suggesting that somatic mutation of the NSDHL contributes to the malignant progression of breast cancer cells. Conclusion: The present data indicate that breast cancer cells harboring somatic mutants of the NSDHL gene seem to display more aggressive behavior by gaining biological worse phenotype both in vitro and in vivo. More investigation is required into the mechanisms behind the function and accumulation of mutant NSDHL proteins to speed the development of therapies for patients with breast cancer harboring mutant NSDHL. Citation Format: Moonjou Baek, Hoe Suk Kim, So-Hyun Yoon, Seungyeon Ryu, Sangeun Lee, Kyoungsook Park, Han-Byoel Lee, Wonshik Han. Overexpression of somatic mutation in NSDHL promotes breast cancer cell migration and tumor growth [abstract]. In: Proceedings of the 2022 San Antonio Breast Cancer Symposium; 2022 Dec 6-10; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2023;83(5 Suppl):Abstract nr P5-13-06.

Abstract P4-09-11: Development of personalized medicine for breast cancer based on elucidation of resistance mechanisms to CDK4/6 inhibitors

Abstract Background CDK4/6 inhibitors are the latest and highly specific CDK inhibitors approved for metastatic and early estrogen receptor (ER)-positive breast cancer patients, improving the survival outcomes. Prior preclinical research has indicated various aberrations of cell cycle regulation and activated oncogenic signaling pathway as the mechanisms of resistance to CDK4/6 inhibitors. However, there is currently no available biomarker except ER and human epidermal growth factor receptor 2 (HER2) in clinical practice. The aim of our study is to elucidate diverse mechanisms of acquired resistance of ER-positive breast cancers to CDK4/6 inhibitors and develop personalized medicine in ER-positive breast cancer patients. Materials and methods To generate resistant lines, the ER-positive human breast cancer cell lines MCF7 and T47D were treated individually with increasing concentrations of the CDK4/6 inhibitors palbociclib and ribociclib until a target concentration of 3μM for six months. Cells were maintained in RPMI 1640 containing 10% fetal bovine serum, 100 U/ml penicillin/streptomycin at 37°C in a 5% CO2 incubator. Western blot analysis was performed for whole cell extract proteins from resistant cells to CDK4/6 inhibitors and parental cells. Colony formation assay was done to observe cell viability. Results Western blot analysis showed elevated FGFR2, CDK6, pS6RP, and cyclin E1 in resistant cells compared to parental cells. Specifically, cyclin E1 and pS6RP were increased in MCF7 palbociclib- and ribociclib-resistant cells, while FGFR2 and CDK6 in T47D palbociclib- and ribociclib-resistant cells. Notably, CDK6 was upregulated in MCF7 ribociclib-resistant cells, whereas increased cyclin E1 was detected in T47D palbociclib-resistant cells, respectively. Colony formation assay revealed acquired ability of resistance to CDK4/6 inhibitors in generated resistant cell lines. Conclusions The tumors harboring resistance to CDK4/6 inhibitors have heterogenous mechanisms of therapeutic resistance to these agents. The novel combination treatment of targeted molecular therapy and CDK4/6 inhibition will lead development of personalized medicine for ER-positive breast cancer based on elucidation of diverse resistant mechanisms to CDK4/6 inhibition. Western blot analysis of lysates from MCF-7 cells treated for 24 to 48 hours with palbociclib (Palbo) and ribociclib (Ribo) Cyclin E1 and pS6RP were increased in MCF7 palbociclib- and ribociclib-resistant cells, whereas CDK6 was upregulated especially in MCF7 ribociclib-resistant cells. Western blot analysis of lysates from T47D cells treated for 24 to 48 hours with palbociclib (Palbo) and ribociclib (Ribo) FGFR2 and CDK6 were increased in T47D palbociclib- and ribociclib-resistant cells, whereas increased cyclin E1 was detected especially in T47D palbociclib-resistant cells. Viability in MCF7 ribociclib-resistant and parental cells by colony formation assay Colony formation assay revealed acquired ability of resistance to CDK4/6 inhibitors in MCF7 resistant-cells. Citation Format: Yoshie Kobayashi, Yusuke Motoi, Mutsumi Fujimoto, Hideo Shigematsu. Development of personalized medicine for breast cancer based on elucidation of resistance mechanisms to CDK4/6 inhibitors [abstract]. In: Proceedings of the 2022 San Antonio Breast Cancer Symposium; 2022 Dec 6-10; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2023;83(5 Suppl):Abstract nr P4-09-11.

Abstract P3-07-26: Anti-cancer activity of naturally formulated Trigonella foenum graecum (Fenugreek) PLGA hyaluronic acid-coated nanoparticles for treatment of breast cancer cell lines

Abstract There are several growing evidences of anticancer complementary and alternative medicines worldwide. Trigonella foenum graecum (Fenugreek) is traditionally applied to treat several disorders such as diabetes, inflammation, and gastrointestinal ailments. Previously, we reported that Fenugreek has anticancer properties against liver cancer due to its active beneficial chemical constituents. Herein, Fenugreek-loaded poly (l-lactic-co-glycolic acid) (PLGA) hyaluronic acid (HA)-coated nanoparticles have been formulated to improve and enhance the anticancer potential of Fenugreek against human breast cancer cell lines. The cytotoxic effects, physicochemical characterization in vitro and pharmacodynamic study of optimized formulation of the FCE-loaded (PLGA)-(HA)-coated nanoparticles in vivo were investigated in (ER-positive) and (triple-negative), breast cancer cell lines, ZR-75-1 and MDA-MB-231, respectively. The particle size, PDI, zeta potential, entrapment efficiency, and loading capacity of FCE-loaded nanoparticles were 188.5 ± 3.1 d.nm, 0.198 ± 0.114, −.118 mV, 38.41 ± 1.4%, and 6.37 ± 1.08%, respectively. The in vitro cytotoxicity study of FCE-loaded NPs by Wst-1 proliferation assay on both tested breast cancer cell lines showed an anti-cancer activity after 48 h of treatment in a dose-dependent manner. Furthermore, the anticancer effect of FCE-loaded nanoparticles was examined by the morphological cell changes, histone release, caspase-3 activity, and modulation of the oncogenic signal pathways of m-tor, β-catenin, cyclin D-1 and STAT3 that are constitutively active in wide variety of breast cancer cell lines which in turn could be important as therapeutic targets in breast cancer. 66 μg/mL FCE-loaded PLGA nanoparticles induced 67.3% apoptosis in both tested cell lines after 48 h that was associated with increased expression of caspase 3, Bax, Bak, p53, and cell cycle arrest at G0/G1 in a time-and dose-dependent manner compared to unloaded-nanoparticles or untreated cells. Moreover, there were significant inhibition in m-tor, β-catenin, cycline-D1, and STAT3 expression levels in both tested cell lines compared to un-loaded-nanoparticles or untreated cells. All in all, our results showed that the formulated FCE-loaded (PLGA)-(HA)-coated nanoparticles induced apoptosis in both ER-positive and triple-negative breast cancer cell lines respectively by increase the expression of pro-apoptotic genes and inhibited several oncogenic genes that might hold a promise for the complementary therapy for human breast cancer. Citation Format: Islam Yahiya, Rana Saeed, Ahmed Sultan. Anti-cancer activity of naturally formulated Trigonella foenum graecum (Fenugreek) PLGA hyaluronic acid-coated nanoparticles for treatment of breast cancer cell lines [abstract]. In: Proceedings of the 2022 San Antonio Breast Cancer Symposium; 2022 Dec 6-10; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2023;83(5 Suppl):Abstract nr P3-07-26.

Inhibitory effect of calycosin on breast cancer cell progression through downregulating lncRNA HOTAIR and downstream targets: HuR and IGF2BP1.

Breast cancer is the most commonly diagnosed cancer worldwide. Previously, we reported that calycosin, a typical isoflavone phytoestrogen, triggers apoptosis and is associated with lncRNA HOTAIR in the estrogen receptor (ER)-positive breast cancer MCF-7-cell line. In the present study, we aim to uncover the mechanism of lncRNA HOTAIR in the inhibitory effect induced by calycosin in both ER-positive and ER-negative breast cancer cell lines. Results show that calycosin significantly inhibits proliferation and triggers apoptosis in both ER-positive (MCF-7 and T47D) and ER-negative (MDA-MB-231 and SK-BR-3) breast cancer cell lines, accompanied by downregulation of lncRNA HOTAIR expression. Accordingly, knockdown of lncRNA HOTAIR promotes the anti-tumor effect of calycosin, while overexpression of lncRNA HOTAIR attenuates this effect. Meanwhile, the expression levels of HuR and IGF2BP1 are also reduced by calycosin. More importantly, calycosin facilitates the downregulation of HuR and IGF2BP1 caused by decreasing lncRNA HOTAIR expression, and the upregulation of HuR and IGF2BP1 caused by overexpression of lncRNA HOTAIR is weakened by calycosin. These results demonstrate that downregulating HuR and IGF2BP1 by suppressing lncRNA HOTAIR results in inhibited growth of breast cancer cells by calycosin. In addition, the binding of HuR and IGF2BP1 to lncRNA HOTAIR is detected by RIP assay, implying an interaction between these two proteins and lncRNA HOTAIR. Together, lncRNA HOTAIR may play a carcinogenic role in breast cancer development and has the potential to be a novel therapeutic target for breast cancer in the future, especially in isoflavone phytoestrogen therapy.

BPTF promotes the progression of distinct subtypes of breast cancer and is a therapeutic target.

To assess the biomarker and functional role of the chromatin remodeling factor, bromodomain PHD finger transcription factor (BPTF), in breast cancer progression. BPTF copy number was assessed using fluorescence in situ hybridization. BPTF expression was regulated in breast cancer cells by shRNA/siRNA-mediated gene silencing and BPTF cDNA overexpression. The effects of regulating BPTF expression were examined on key oncogenic signaling pathways and on breast cancer cell proliferation, apoptosis, and cell cycle progression, as well as in xenograft models. The consequences of pharmacological bromodomain inhibition, alone or in combination with other targeted agents, on breast cancer progression were assessed in culture and in xenograft models. BPTF copy number was gained in 34.1% and separately amplified in 8.2% of a breast cancer tissue cohort. Elevated BPTF copy number was significantly associated with increasing patient age and tumor grade and observed in both ER-positive and triple-negative breast cancer (TNBC) subtypes. BPTF copy number gain and amplification were also observed in The Cancer Genome Atlas (TCGA) breast cancer cohort. Stable shRNA-mediated silencing of BPTF significantly inhibited cell proliferation and induced apoptosis in TNBC and ER-positive human breast cancer cell lines. BPTF knockdown suppressed signaling through the phosphoinositide 3 kinase (PI3K) pathway, including reduced expression of phosphorylated AKT (Ser473), phosphorylated GSK-β (Ser9), and CCND1. These findings were confirmed following transient BPTF knockdown by a distinct siRNA in TNBC and ER-positive breast cancer cells. Stable suppression of BPTF expression significantly inhibited the in vivo growth of TNBC cells. Conversely, BPTF cDNA overexpression in TNBC and ER-positive breast cancer cells enhanced breast cancer cell proliferation and reduced apoptosis. BPTF targeting with the bromodomain inhibitor bromosporine, alone or in combination with the PI3K pathway inhibitor gedatolisib, produced significant anti-tumor effects against TNBC cells in vitro and in vivo. These studies demonstrate BPTF activation in distinct breast cancer subtypes, identify pathways by which BPTF promotes breast cancer progression, and suggest BPTF as a rational target for breast cancer therapy.

Abstract 424: Development of HJC0152-based proteolysis-targeting chimera degraders for breast cancer therapy

Abstract Signal transducer and activator of transcription 3 (STAT3), a member of the STAT family, is constitutively activated in various human cancers, and has been recognized as an attractive therapeutic target in cancer. STAT3 protein dimerizes and translocates to the nucleus to eventually transactivate downstream target genes. Despite some progress has been made in the past two decades, to date there is still lack of FDA-approved STAT3 inhibitors available for clinical use. Recently, proteolysis-targeting chimera (PROTAC) technology has dramatically advanced the development of therapeutics by introducing targeted protein degradation. Previously, our team has developed HJC0152, a promising STAT3 inhibitor, via structure- and fragment-based drug design strategies and in silico molecular modeling and docking. HJC0152 has demonstrated an attractive cancer therapeutic and preventive efficacy by targeting the non- estrogen receptor (ER)-based STAT3 signaling pathway. To achieve better potency and inhibition of STAT3 signaling, we therefore developed a series of HJC0152-based PROTAC protein degraders. These potential anticancer agents have been initially evaluated and several showed favorable potency in inhibiting the proliferation in a panel of breast cancer cell lines. We found that compounds JMX1122, JMX1123, and JMX1124 significantly inhibited proliferation and colony formation of a highly aggressive triple-negative breast cancer (TNBC) cell line, MDA-MB-231, and the ER-positive breast cancer cell lines. Different from the lead compound HJC0152, which showed an early inhibition on the phosphorylation of STAT3 at the Tyr705 and Ser727 residues, the newly developed HJC0152-PROTACs showed a significantly late differential inhibition of STAT3 phosphorylation at the Tyr705 and Ser727 residues, and reduced the total STAT3 protein level at 72 hours of treatment. Our current results support the notion that HJC0152-based PROTAC degraders potently suppress breast cancer cell proliferation and induce apoptosis, via direct targeting of STAT3 and its protein degradation. These compounds are currently being investigated for breast cancer cell migration and invasion, and will be evaluated for the in vivo inhibition of tumor growth and metastasis in different mouse models. This project is supported by NIH/NCI Awards R01CA226001 and R01CA231150 to Q.S. and J.Z. Citation Format: Junhai Xu, Jimin Xu, Ruixia Ma, Mingxiang Zhou, Doerte R. Fricke, Haiying Chen, Hyejin Kim, Xi Liu, Jia Zhou, Qiang Shen. Development of HJC0152-based proteolysis-targeting chimera degraders for breast cancer therapy [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 424.

Abstract 1777: Androgen receptor (AR) agonists inhibit AR- and estrogen-receptor-positive breast cancer

Abstract Introduction: Breast cancer is the most common cancer diagnosed in women. About 13% of women will develop invasive breast carcinoma in their lifetime and it is estimated that about 280,000 new cases will be diagnosed in 2021. Seventy-percent of diagnosed breast cancers are estrogen receptor (ER)-positive and about 90% of the ER-positive breast cancers are androgen receptor (AR)-positive. We have previously shown that AR agonists inhibit ER-positive breast cancer growth by sequestering the pioneer-transcription factor FOXA1 from ER-cistrome. Tissue-selective AR modulators (SARMs) such as enobosarm are in clinical trials to treat ER-positive breast cancers. Since resistance is a major impediment to sutained treatment, it is important to identify new therapeutic strategies and their mechanisms of resistance. Here, we evaluated the possible mechanisms of resistance to AR agonists in ER-positive breast cancer. Description: ER-positive breast cancer cell line xenografts and patient-derived xenografts (PDX) were used as experimental models. Once tumors grew to ~100-300 mm3, mice were randomized and treated orally with vehicle or enobosarm (30 mg/kg/day). Animals were sacrificed after 4 weeks when the tumors responded to treatment and after 10 weeks once resistance developed to treatment. Tumor volumes were measured twice weekly and tumors were collected at sacrifice for further analyses. RNA-seq and ChIP-seq were performed on tumor tissues. Summary: AR agonists, enobosarm and dihydrotestosterone (DHT), inhibited the proliferation of ER-positive breast cancer cells ZR-75-1 and T47D. Transcriptome analysis revealed that AR agonists activate AR in ER-positive breast cancer cells and inhibit ER-target gene signature. AR agonists inhibited the growth of breast cancer cell line xenograft T47D and breast cancer PDXs HCI-7 that express wildtype ER, and HCI-13 and WHIM-23, two models that express Y537S mutant ER. Treatment of WHIM-23 and T47D xenograft for over ten weeks resulted in resistance and regrowth. AR agonist-sensitive and -resistant tumors were analyzed using RNA-seq, ChIP-seq, and ATAC-seq to understand the mechanism of resistance development. The results indicate that the prolonged activation of AR will result in resistance. These mechanisms of resistance can be utilized as possible therapeutic targets. Conclusion: These results suggest that the AR is a promising therapeutic target in the treatment of ER-positive breast cancer. However, resistance development is possible with AR agonists and the mechanism provides suggestions for future combination therapies. Disclosure: This work was supported by an NCI grant (CA229164 and CA229164S1 to RN) and by a DOD grant (W81XWH2110055 to RN). Citation Format: Sarah Asemota, Suriyan Ponnusamy, Thirumagal Thiyagarajan, Ramesh Narayanan. Androgen receptor (AR) agonists inhibit AR- and estrogen-receptor-positive breast cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 1777.

The Effects of Deoxyelephantopin on the Akt/mTOR/P70S6K Signaling Pathway in MCF-7 Breast Carcinoma Cells In Vitro

Objective To determine the effects of deoxyelephantopin on mTOR and its related target molecules (Akt/mTOR/P70S6K) in the ER-positive breast cancer cell line. Materials and Methods Primary in silico simulations were determined, and the effects of deoxyelephantopin on the phosphorylation of the Akt/mTOR/P70S6K molecules were evaluated using AlphaScreen-based assays and western blot analysis, respectively. Results Based on the estimated FEB and K i values, deoxyelephantopin appeared to have a stronger affinity toward P70S6K as compared with Akt and mTOR. Both deoxyelephantopin and control inhibitors were observed to form hydrogen bonds with the same key residue, Leu175 of the P70S6K molecule. Deoxyelephantopin downregulated the p-P70S6K protein expression significantly from 18 µM ( p &lt; 0.05) and onward. Based on the AlphaScreen assay, deoxyelephantopin produced a concentration-dependent inhibition on the phosphorylation of P70S6K with an IC50 value of 7.13 µM. Conclusion Deoxyelephantopin induced cell death in MCF-7 cells possibly via DNA fragmentation, inhibition of the phosphorylation of P70SK6, and downregulation of the relative p-p70S6K protein expression levels.

Structure-activity relationship studies on O-alkylamino-tethered salicylamide derivatives with various amino acid linkers as potent anticancer agents

In our continued SAR study efforts, a series of O-alkylamino-tethered salicylamide derivatives with various amino acid linkers has been designed, synthesized, and biologically evaluated as potent anticancer agents. Five selected compounds with different representative chemical structures were found to show broad anti-proliferative activities, effective against all tested ER-positive breast cancer (BC) and triple-negative breast cancer (TNBC) cell lines with low micromolar IC50 values. Among these compounds, compound 9a (JMX0293) maintained good potency against MDA-MB-231 cell line (IC50 = 3.38 ± 0.37 μM) while exhibiting very low toxicity against human non-tumorigenic breast epithelial cell line MCF-10A (IC50 > 60 μM). Further mechanistic studies showed that compound 9a could inhibit STAT3 phosphorylation and contribute to apoptosis in TNBC MDA-MB-231 cells. More importantly, compound 9a significantly suppressed MDA-MB-231 xenograft tumor growth in vivo without significant toxicity, indicating its great potential as a promising anticancer drug candidate for further clinical development.

Abstract P5-06-12: Extracellular adenosine synthesis genes regulated by estrogen signaling are associated with cancer aggressiveness and poor prognosis in estrogen receptor (ER)-positive breast cancer

Abstract Background: Accumulation of extracellular adenosine regulates tumor progression. Extracellular adenosine binds to adenosine receptors, which mediates signaling to induce angiogenesis and cell proliferation, as well as functioning as an immunosuppressive agent in the tumor microenvironment (TME). CD73 and CD39 are two cell surface enzymes that catalyze the synthesis of extracellular adenosine from AMP, ADP and ATP in the TME. However, the underlying mechanisms that regulate adenosine synthesis in the TME of ER-positive breast cancer remains unknown. Methods: In order to investigate the transcriptional regulation of CD73 and CD39 in ER-positive breast cancer, we treated ER-positive breast cancer cell line, MCF7 with estrogen and tamoxifen. We also investigated the clinical significance of CD73/39 expression in ER-positive patients by bioinformatical approach using TCGA and GEO breast cancer cohorts. Results: In TCGA cohort, higher CD73 expression was associated with worse overall survival in ER-positive tumors (p=0.003), but not in ER-negative tumors. Gene set enrichment analysis revealed that estrogen response gene sets (Early; p=0.043, Late; p=0.021) were significantly enriched in CD73 low expressing ER-positive tumors, suggesting estrogen signaling may inhibit CD73 expression. To test this hypothesis, we analyzed the expression of CD73 and CD39 in MCF7 cells treated with estrogen, tamoxifen, or vehicle control. Our data revealed that estrogen treatment suppressed CD73 and CD39 expression, however tamoxifen treatment significantly enhanced expression of the genes. These findings suggest that tamoxifen treatment can induce the expression of CD73 and CD39 in ER-positive breast tumors, by removing the repressive effect of hormone signaling. Additionally, gene set enrichment analysis revealed that CD73-high ER-positive tumor was significantly associated with cancer aggressiveness characteristics, such as epithelial-mesenchymal transition (EMT) (p&amp;lt;0.001) and angiogenesis (p&amp;lt;0.001). On the other hand, CD73-high ER-positive tumor have significantly less infiltrating CD8-positive T cells, memory B cells and plasma cells in silico analysis, implying that CD73-high tumors have immunosuppressive environment. Further, we found that CD73 expression was significantly elevated post-chemotherapy as compared to the tumors prior to the treatment (p=0.007), and CD73 high expressing patients demonstrated worse relapse-free survival in the neoadjuvant chemotherapy cohort (p=0.003). Conclusion: Our molecular findings indicate that expression of CD73 and CD39 are transcriptionally repressed by estrogen signaling, however tamoxifen treatment reverses the effect. Increased expression of CD73 significantly correlates with worse outcomes in ER-positive breast cancer patients which may be due to immunosuppressive tumor microenvironment created by extracellular adenosine driving a pro-metastatic phenotype. Our data indicate an intriguing mechanism which could be therapeutically exploited by targeting CD73/39 to reverse ‘immune-cold’ microenvironment for the treatment of recurrent and metastatic ER-positive breast cancers. Citation Format: Eriko Katsuta, Tao Dai, Abhisha Sawant Dessai, Subhamoy Dasgupta. Extracellular adenosine synthesis genes regulated by estrogen signaling are associated with cancer aggressiveness and poor prognosis in estrogen receptor (ER)-positive breast cancer [abstract]. In: Proceedings of the 2021 San Antonio Breast Cancer Symposium; 2021 Dec 7-10; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2022;82(4 Suppl):Abstract nr P5-06-12.